Stevia plant named `RSIT 95-166-13`

ABSTRACT

A novel Stevia rebaudiana Bertoni variety characterized by a high ratio of rebaudioside C to stevioside, a high ratio of rebaudioside C to rebaudioside A, and a high ratio of rebaudioside C to dulcoside A in its leaves.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinct variety of stevia(Stevia rebaudiana Bertoni), referred to by the varietal name `RSIT95-166-13`.

Eight steviol glycosides with sweetening properties have been identifiedin leaf tissues of stevia. The four major glycosides are: stevioside,rebaudioside A, rebaudioside C, and dulcoside A. The chemical structuresof these compounds are shown in FIG. 1. Each of these steviol glycosideshas unique sensory properties, and they can be used singly or incombination to provide a sweetener that has sensory properties tailoredto a specific use. Stevia sweeteners are non-caloric, making themsuitable for diabetics and weight conscious consumers, and are pH-stableand heat-stable, making them useful in a wide range of bottledbeverages, confectioneries, baked goods, and dairy and canned products.For reviews of the use of stevia as a source of sweteners, see, e.g.,Handro and Ferreira, 1989, Lee 1982, and Phillips, 1987.

We set out to develop stevia varieties with high concentrations ofindividual steviol glycosides that could be extracted and recombined inratios suitable for specific product uses. Landrace stevia has acombination of steviol glycosides that is not optimal for all productapplications.

We discovered our new variety in a cultivated area at the Agricultureand Agri-Food Canada Research Station near Delhi, Ontario, Canada("Delhi Research Station"). The following unique combination ofcharacteristics is outstanding in `RSIT 95-166-13` and distinguishes itfrom its parents and all other stevia varieties of which we are aware:(1) a high ratio of rebaudioside C to stevioside; (2) a high ratio ofrebaudioside C to rebaudioside A; and (3) a high ratio of rebaudioside Cto dulcoside A.

This variety has not been observed under all possible environmentalconditions. The following observations, measurements and comparisonsdescribe plants grown under conditions that are similar to thosegenerally used in commercial practice.

Asexual reproduction of this new variety by shoot-tip and stem cuttingswas performed at the Delhi Research Station and showed that theforegoing characteristics are established and transmitted throughsucceeding asexual propagations.

BRIEF DESCRIPTION OF THE PHOTOGRAPHS

The accompanying photographs show typical specimens of this new variety,depicted in color as nearly true as is reasonably possible in a colorphotograph of this character. The photographs were taken at the DelhiResearch Station in February 1996.

FIG. 1 shows the chemical structure of four major steviol glycosidesweeteners in stevia, stevioside, rebaudioside A, rebaudioside C, anddulcoside A.

FIG. 2 is a view of a plant of the present invention.

FIG. 3 is an enlarged view of leaves of the plant of FIG. 2.

DETAILED DESCRIPTION

In the summer of 1994, ten plants from different seed germplasmaccession of stevia from the People's Republic of China were grown forevaluation at the Delhi Research Station. Plant samples `RSIT 94-1838`,`RSIT 94-1833`, and `RSIT 94-1560` were retained as a result of thehigher than average concentrations of rebaudioside C on a dry weightbasis in their leaves (Table 1). A plant (`RSIT 94-1829`) selected fromthe variety `Brazil Zairai` was also retained, since its leaves hadhigher than average rebaudioside C concentrations (Table 1). `BrazilZairai` is an open-pollinated landrace variety of stevia obtained fromthe Japanese National Germplasm Depository.

These four clones were used as parents and intercrossed in the fall andwinter of 1994-1995. Half sib seeds were collected from clone, `RSIT94-1560` and were designated half sib population `RSIT 95-166`. Plantsfrom half sib population `RSIT 95-166` were evaluated in the field inthe summer of 1995 and one plant, `RSIT 95-166-13`, was selected on thebasis of its novel steviol glycoside traits. See Brandle and Rosa (1992)for additional information regarding growth conditions and sexualcrosses of Stevia varieties.

In trials conducted in 1994-95, the steviol glycoside profiles in leavesof the following stevia varieties were compared: `RSIT 95-166-13`; `RSIT94-1560`, the maternal parent used to produce the half sib populationfrom which `RSIT 95-166-13` was selected; `RSIT 94-1838`, `RSIT94-1833`, and `RSIT 94-1829`, the paternal parents used to produce thehalf sib population from which RSIT 95-166-13 was selected; and`Brazil`. In particular, the following were examined (on a dry weightbasis): percent dulcoside A (% dulc.), percent stevioside (% stev.),percent rebaudioside C (% reb.C), percent rebaudioside A (% reb.A), andpercent total glycosides (% total=% dulc.+% stev.+% reb.C+% reb.A). Theresults of the comparison are shown in Table 1.

`RSIT 95-16613` was dug out of the field in the fall of 1995 andvegetatively propagated at the Delhi Research Station by shoot-tip andstem cuttings.

                  TABLE 1                                                         ______________________________________                                        Steviol Glycoside Profiles of `RSIT 95-166013`, `RSIT                         94-1560`, `RSIT 94-1838`, `RSIT 94-1833`,                                     `RSIT 94-1829`, and `Brazil`                                                                  %       %    %      %     %                                   Variety Year    dulc.   stev.                                                                              reb. C reb. A                                                                              Total                               ______________________________________                                        95-166-13                                                                             1995a.sup.1                                                                           0.47    0.30 14.40  0.37  15.55                               95-166-13                                                                             1995b.sup.2                                                                           0.82    0.36 14.78  0.44  16.41                               94-1560 1995    0.64    3.78 5.59   3.70  13.71                               94-1560 1994    0.91    4.9  7.2    4.9   17.91                               94-1838 1994    0.96    4.4  10.0   5.7   21.06                               94-1833 1994    1.19    4.8  7.4    4.3   17.69                               91-1829 1994    1.75    7.2  6.9    4.6   20.45                               Brazil  1995    0.25    7.61 0.54   3.00  11.40                               ______________________________________                                         .sup.1 1995a was sampled in August 1995.                                      .sup.2 1995b was sampled in September 1995.                              

Taxonomic Description of `RSIT 95-166-13`

`RSIT 95-166-13` is a suffrutescent, erect perennial (FIG. 2).Field-grown plants may attain a height of 1.1 meters, but more commonlyhave a height of 6-8 dm. The stems are round, pubescent, and haveinternodes of medium length. The leaves are simple, opposite, sessile,exstipulate, and oblanceolate. The leaf apices are obtuse and the leafmargins are crenate above the middle and entire on a cuneately narrowbase. The leaves are three-nerved and conspicuously veiny. The leafsurface is puberulent with short glandless hairs. The largest caulineleaves are up to 7 cm long and 3 cm wide and are often proliferous inthe axils (FIG. 3).

The inflorescence is loosely paniculate with the heads appearingopposite the bracts in irregular sympodial cymes. The flower corollashave a pale purple throat and white limb. The seed are nearly uniformachenes, 15-17 aristate (Robinson, 1930).

Method of Determining Steviol Glycosides and Concentrations

Fresh leaf samples were harvested from field-grown stevia plants andstored in a freezer prior to extraction. Samples were freeze dried,ground to pass through a 40-mesh screen, and again freeze dried beforeextraction. Ground, freeze-dried plant material (300 mg.) was weighedinto a 15 ml (polypropylene centrifuge tube (Fisher). Ten ml of 1:1(v/v) acetonitrile-water (acetonitrile from Caledon Laboratories Ltd.,Georgetown, Ontario, 190 HPLC grade) was added to each sample. Sampletubes were suspended in an ultrasonic bath (Lab-line Inst. model #9333,Melrose Park, IL) at maximum ultrasonication (#9) for 15 min withoccasional stirring and rotation to ensure optimal extraction. Sampletubes were transferred to a centrifuge (International Centrifuge, ModelV Size 2, International Equipment, Boston, MA) and centrifuged at 1500rpm for 15 min. The extraction solvent was transferred to a volumetricflask with a Pasteur pipette. For routine analyses, duplicateextractions were utilized and the solvent transferred to a 25 mlvolumetric flask filled to volume with acetonitrile. Samples werefiltered through a 0.45 micron filter (Acrodisc 13, Gelman Sciences, AnnArbor, MI).

Analyses were performed on a liquid chromatograph (Hewlett-Packard 1090)equipped with a three-channel solvent delivery system, auto sampler, anddiode array detector interfaced with a chem station. Stevioside,rebaudioside A, rebaudioside C, and dulcoside A standards were suppliedby FWB Chemical Consulting Ltd., Calgary, Alberta, Canada. Steviosidewas also obtained from Sigma Chemical Co., St. Louis, Mo. Due to thewide variation in plant material analyzed, co-eluting peaks occasionallywere apparent in the chromatogram. Often these plant constituents wereeliminated by filtering them through a Waters NH2 Sep-Pak cartridge(Part No. WATO 20535) prior to analysis. For routine analyses, onlystevioside was used as a standard. The response factor found forstevioside was used for the other three steviol glycosides aftercorrecting for differences in molecular weight. In order to monitor thisassumption during the sample determinations, a standard sample ofrebaudioside A was analyzed after each calibration.

The chromatographic column was a Waters cartridge carbohydrate column(Part No. WAT 044355) having an inside diameter of 250×4.6 mm. The guardcolumn was a carbohydrate sentry guard column (Part No. WAT 046895). The"A" solvent was H₂ O (pH 5.25 with AcOH), the "B" solvent wasacetonitrile, and the "C" solvent was acetonitrile. The flow rate was1.5 ml/min. The time table for use of solvents A, B, and C is found inTable 2.

                  TABLE 2                                                         ______________________________________                                        Time table for use of Solvent A (H.sub.2 O, pH 5.25 with AcOH),               Solvent B (acetonitrile), and Solvent C (acetonitrile)                        Time (min.)                                                                             % Solvent A  % Solvent B                                                                             % Solvent C                                  ______________________________________                                        0         13           43        44                                           12        17.5         41        41.5                                         26        24           38        39                                           26.1      13           43        44                                           ______________________________________                                    

The post time was six minutes and the elution times for the steviolglycosides were 14.5 minutes for dulcoside A, 18.8 minutes forstevioside, 20.4 minutes for rebaudioside C, and 23.5 minutes forrebaudioside A. Retention times of the components of interest decreasedwith extensive use of the column. It was usually sufficient to simplyreduce the amount of water in the initial chromatographic conditions torestore the retention times. Individual columns have been used for morethan one thousand samples. Guard columns typically were replaced afterabout 300 samples or when system pressure became excessive. Periodicallythe column was washed with water to remove other plant constituentswhich accumulated on the column. Steviol glycosides were identified byretention time and concentrations were determined by an externalstandards method.

Steviol glycosides may be produced from leaves or other parts of `RSIT95-166-13` and formulated into a sweetener for use in the food industryor by consumers by any conventional method. See, e.g., U.S. Pat. No.4,892,938. Purified extract solutions may be directly added to afoodstuff or beverage, for example, in liquid form or converted to acrystalline form. The sweetness intensity of steviol glycosides isusually about 150 to 300 times that of sucrose, depending on theconcentration of the steviol glycosides used. If desired, steviaextracts can be suitably diluted with a conventional diluent, e.g., atasteless soluble starch, to achieve a sweetness intensity comparable tothat of sugar (i.e., sucrose). For convenient use by the consumer, aunit of a stevia sweetener, e.g., an amount having a total sweetnessapproximately that of a standard lump of sugar, can be packaged forcommercial distribution in small paper bags or in other forms known inthe art. Steviol glycosides from `RSIT 95-166-13` can be used singly orin combination with steviol glycosides from other stevia varieties orwith other conventional sweeteners to provide a sweetener compositionthat has sensory properties tailored to a specific use.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Reference Cited

Brandle, J., Rosa, N. (1992). "Heritability for Yield, Leaf:Stem Ratioand Stevioside Content Estimated from a Landrace Cultivar of Steviarebaudiana." Can. J. Plant Sci. 72: 1263-1266.

Handro, W., Ferreira, C.M. (1989). "Stevia rebaudiana (Bert.) Bertoni:Production of Natural Sweeteners." In: Biotechnology in Agriculture andForestry, vol. 7, Medicinal and Aromatic Plants II (ed. Y.P.S. Bajaj),Springer Verlag, Berlin, Heidelberg, pp. 468-487.

Lee, J.I., et al. (1982). "New High Rebaudioside-A Stevia Variety`Suweon 11`." Res. Rept. ORD 24(C):186-188.

Phillips, K.C. (1987). "Stevia: Steps in Developing a New Sweetener."In: Developments in Sweeteners-3, Elsevier Applied Science, London (ed:T.H. Grenby). pp. 1-43.

Robinson, R.L. (1930). Contributions from the Gray Herbarium of HarvardUniversity XC, The Gray Herbarium of Harvard University, Cambridge,Mass. pp. 78-91.

U.S. Pat. No. 4,892,938 (1990).

We claim:
 1. A new and distinctive variety of Stevia rebaudiana Bertoniplant, substantially as herein shown and described.